Alterations in retinal tau phosphorylation in Alzheimer's disease patients identified by mass spectrometry
Background: Most neurodegenerative diseases, including Alzheimer's disease (AD) and multiple sclerosis (MS), are associated with abnormal post-translational phosphorylation of tau (p-tau) in the brain. Studies using immunostaining techniques have revealed an accumulation of p-tau also in the AD retina and suggested this p-tau accumulation may reflect tau pathology in the brain. However, immunostainings are dependent on antibody specificity and tissue processing. Hence, further validation using additional methods is needed to identify the p-tau species in the retina and verify their relationship to AD pathology. Methods: We used mass spectrometry to measure p-tau peptides in retinal and hippocampal samples from non demented controls (NC, n=8), AD ( n=12), and MS (n=4). Peptides were first extracted with Lysis buffer to capture extracellular components and cytoplasm, and then with Ripa buffer to isolate nuclear and organelle proteins. We then analysed the differences in p-tau levels between diagnoses and explored how retinal p-tau variants correlate with hippocampal p-tau and neuropathological changes. Results: The mass spectrometry analysis of the retina revealed peaks corresponding to tau peptides phosphorylated at T181, S199/S202, T231, S396 + T403/S404, and T403/S404. These p-tau peptides were also detected in the hippocampal samples, along with additional p-tau peptides such as T217 and S262. Total tau phosphorylation and phosphorylation at S199/S202 and T231 were significantly higher in the retina of AD cases compared to NC. These two peptides, along with peptides phosphorylated at S396+T403/S404 and T403/S404, were also higher in cases with high amyloid-beta (A{beta}) Braak stages compared to those with low A{beta} Braak stages. Further analysis showed that higher A{beta} Braak stages were associated with higher mass spectrometry peak intensities of peptides phosphorylated at S199/S202 and S396+T403/S404. Additionally, retinal p-tau peptides at S396+T403/S404 and T403/S404 2 correlated with neurofibrillary tangle (NFT) Braak stages, and p-tau peptides S396+T403/S404 in the retina were linked to corresponding phosphorylaion in the CA1 region. Conclusions: These findings underscore the connection between retinal and brain tau pathology and highlight the potential of retinal tau as a biomarker for AD diagnosis and monitoring while also deepening our understanding of tauopathies in both the retina and brain.